1. Field of the Invention
The present invention relates to liver parenchymal cells having a clonal growth ability, a method for obtaining such cells, a method for subculturing such cells, and a subculturing system of primary hepatocytes. More particularly, the present invention relates to progenitor cells culturing system of liver parenchymal cells which are useful as a material for cell biological and molecular biological research on development, differentiation and proliferation process of hepatocytes or on the carcinogenic mechanism thereof, or as medical materials for developing therapeutic techniques of various hepatic diseases.
2. Description of Related Art
An animal is a multicellular organism formed through repeated division of a fertilized egg and differentiation thereof into various structures (cell aggregates) taking charge of different functions. The individual structures composing a body of organism maintain the individual by producing cells having active differentiation ability through constant division and growth of individual cells. Therefore, in order to understand the biological facts of humans and other animals or to develop a therapeutic technique through clarification of the carcinogenic mechanism, it is believed important to analyze in detail cells composing individual structures to clarify the developing and differentiating process and the mechanism of proliferation.
A method has conventionally been established, as a means to analyze in detail cells of structures in vivo, to culture cells taken out in vitro, and causing division and growth of cultured cells to ensure survival through subcultures. While various methods of subculturing primary hepatocytes have been studied, the only actual case of subculture of primary hepatocytes so far reported is one for calf, and no case has yet been achieved as to rat and mouse. A reason is that, because hepatocytes are cultured in a serum-free medium added with various growth factors on a collagen-coated dish effective for adhesion and growth of hepatocytes, it is difficult to detach the cultured cells from the dish with a little damage, and hepatocytes treated with an enzyme such as trypsin have very serious damage. While hepatocytes cultured with a dish not coated with collagen coat and a serum-free medium can be detached with a slight damage, those cultured cells cannot continue to live or grow for a long period of time, although it may be possible to adhere them again onto the dish.
More recently, on the other hand, it has been reported that small hepatocytes growing while forming a colony appear in a culture system comprising a medium added with nicotinamide and epidermal growth factor (EGF). These small hepatocytes are confirmed to express functions of matured hepatocytes such as albumin, to be divided 2 to 3 times during the first four days, and to be partially present on the 20th day of culture in the form of cells having a growth ability. Since the colony forming frequency in such a culture system is high for hepatocytes isolated from an young rat and decreases according as the age in weeks increases, these subcultured hepatocytes are considered to be "committed progenitor cells".
In this case also, however, subculturing of cells is not satisfactory, leaving problems to be solved regarding prevention of damage. It is therefore the current situation the subculturing of hepatocytes has not as yet been established.
Furthermore, in order to understand complicated and diverse functions of hepatocytes or clarify carcinogenic mechanism thereof, it is considered essential to identify pure precursor hepatocytes (progenitor cells) for which orientation of differentiation has not yet been specified, but presence thereof has not yet been confirmed or a preferential culturing method has not been established.
For these progenitor cells, the following facts are conventionally known and the following efforts to identify them are reported. More specifically, it is reported that stem cells are developed from the foregut endoderm in the course of liver development, and these stem cells differentiate into hepatocytes and bile duct epidermal cells (Shiojiri, et al.; Cancer Research. Vol. 51, pp. 2611-2620, 1991). While there is no case of confirmation of stem cells in liver of an adult (such as rat), oval cells emerging in a precancerous state in the course toward cancer of rat can lead to either hepatocelluler carcinoma or cholangiocarcinoma. This oval cell is therefore attributable to an aberrant differentiation of stem cells present in the liver of the adult rat. Hixon, et al.(Pathobiology, Vol. 58, pp. 65-77, 1990) prepared several antibodies against surface antigens of oval cells obtained from experimental hepatocarcinogenesis. Brill, at al.(Proc. Soc. Exp. Bio. MEd., Vol. 204, pp. 261-269, 1993) selected cells conbined with these antibodies from among hepatocytes of an adult rat by means of a sorter to investigate properties of these cells. As a result, it was suggested that these cells contained hepatic progenitor cells among those combined with antibodies against surface antigens of oval cells, since cells growing and differentiating into matured hepatic cells by culturing in a medium added with additive factors, or culturing on a feeder layer of mesenchyme cells of a fetus.
Presence of hepatic progenitor cells is estimated by several pieces of evidence, but has not as yet been confirmed.